CDC34 Human, His
Description
Production and Purification
Expression System:
Formulation:
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Sterile solution containing 1 mg/mL protein in 20 mM Tris-HCl (pH 8.0), 10% glycerol, 50 mM NaCl, and 1 mM DTT .
Purity:
Functional Insights
Role in Ubiquitination:
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Catalyzes Lys-48-linked polyubiquitination of substrates like IκBα and p27/Kip1 in conjunction with SCF E3 ligases .
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Facilitates cell cycle progression by degrading cyclin-dependent kinase inhibitors (e.g., Sic1 in yeast) .
Mechanistic Features:
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Dimerization: N-terminal fusion tags (e.g., GST or FKBP) induce dimerization, enhancing ubiquitin chain assembly independent of SCF .
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C-terminal ubiquitin binding: UBS1 aromatic residues (Tyr-210, Tyr-211) orient donor ubiquitin for SCF-dependent conjugation .
Biochemical Properties
Stability:
Activity Assays:
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Ubiquitination assays: Demonstrated using reconstituted SCF complexes (e.g., SCFβ-TrCP2) and substrates like IκBα .
Binding Affinities:
| Interaction Partner | Binding Region | Affinity (K<sub>d</sub>) |
|---|---|---|
| Ubiquitin (monomer) | UBS1 | 0.2–0.5 mM |
| Ubiquitin (Lys-48-linked di-Ub) | UBS1 | 0.049 mM |
Research Findings
C-Terminal Ubiquitin Binding:
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NMR studies identified UBS1 and UBS2 as non-covalent ubiquitin-binding motifs .
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Mutations in UBS1 (e.g., Y210G/Y211G) impair SCF-mediated ubiquitination by 70–80% .
SCF Dependency:
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Neddylated ROC1-CUL1 complex enhances CDC34 activity by 5-fold, enabling processive polyubiquitination .
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Disruption of UBS1 abolishes SCF stimulation but preserves basal E2 activity .
Yeast Complementation:
Applications
Product Specs
SPPAFRFLTK MWHPNIYETG DVCISILHPP VDDPQSGELP SERWNPTQNV RTILLSVISL LNEPNTFSPA NVDASVMYRK WKESKGKDRE
YTDIIRKQVL GTKVDAERDG VKVPTTLAEY CVKTKAPAPD EGSDLFYDDY YEDGEVEEEA DSCFGDDEDD SGTEES.
Product Science Overview
The human recombinant CDC34 protein is typically produced in Escherichia coli (E. coli) and is fused to a 20 amino acid N-terminal His-Tag. This recombinant protein is a single, non-glycosylated polypeptide chain containing 256 amino acids, with a molecular mass of approximately 28.9 kDa . The His-Tag facilitates purification through affinity chromatography techniques.
CDC34 is essential for the ubiquitin-mediated degradation of cell cycle G1 regulators and the initiation of DNA replication. It accepts ubiquitin from the E1 complex and catalyzes its covalent attachment to target proteins. This process is crucial for the regulation of various cellular processes, including cell division, signal transduction, and development .
CDC34 works in association with different E3 complexes, such as the SCF (Skp, Cullin, F-box containing complex), to target substrates for ubiquitination and subsequent degradation. Some of the well-characterized substrates of CDC34 include IκB, B-Myb, Wee1, MyoD, ICERIIγ, ATF5, p27Xic1, and p27Kip1 . Additionally, substrates like β-catenin, p21Cip1, E2F, cyclin E, and cyclin D are putative targets of CDC34 due to their requirement for SCF-mediated proteolysis .
The recombinant CDC34 protein is widely used in in vitro ubiquitination reactions to study the ubiquitin-proteasome pathway and its role in various cellular processes. Its ability to self-associate through a domain in the C-terminus and its phosphorylation and ubiquitinylation in vivo make it a valuable tool for research .
The CDC34 protein solution is typically formulated with 20mM Tris-HCl (pH 8), 10% glycerol, 50mM NaCl, and 1mM DTT. It should be stored at 4°C if used within 2-4 weeks or frozen at -20°C for longer periods. For long-term storage, adding a carrier protein (0.1% HSA or BSA) is recommended to avoid multiple freeze-thaw cycles .
